Journal: Clinical Cancer Research

Article Title: SHP2 Is Overexpressed and Inhibits pSTAT1-Mediated APM Component Expression, T-cell Attracting Chemokine Secretion, and CTL Recognition in Head and Neck Cancer Cells

doi: 10.1158/1078-0432.ccr-12-1517

Figure Lengend Snippet: Figure 2. SHP2 depletion upregulates pSTAT1, pJAK-1/2, and total STAT1. (A–F) PCI-13 and (G–L) SCC-90 cells were transfected with SHP2 siRNA (100 nmol/L, 48 hours), which significantly reduced SHP2 expression in HNC cells by Western blot analyses. SHP2 depletion also significantly upregulated pSTAT1 and its target gene product STAT1 as determined by intracellular flow cytometry and immunblotting compared with control siRNA in (B, C, E) PCI-13 and (H, I, K) SCC-90 cells. SHP2 depletion also upregulated pJAK1 (Tyr1022/1023) and pJAK2 (Tyr 1007/1008) compared with control siRNA in (F) PCI-13 and (L) SCC-90 cells. The cells were treated with IFN-g (100 U/mL ¼ 1,464 pg/mL, 48 hours) as a positive control for STAT1 phosphorylation. Data represent 3 independent experiments and error bars indicate SE.

Article Snippet: Anti-pSTAT1 Tyr701 mAb (Cell Signaling Tech), anti-total STAT1 (C-24) polyclonal (pAb; Santa Cruz Biotech), anti-b-actin mAb (Sigma-Aldrich Inc.), anti-rabbit IgG-HRP (Promega), anti-mouse IgG-HRP (Bio-Rad), anti-phosphorylated JAK-1 (Tyr1022/1023) mAb, antiphosphorylated JAK-2 (Tyr 1007/1008) mAb (Cell signaling Tech), anti-total JAK-1 mAb, and anti-total JAK-2 mAb (Santa Cruz Biotech) were used in immunoblot analyses.

Techniques: Transfection, Expressing, Western Blot, Cytometry, Control, Positive Control, Phospho-proteomics

Journal: Cancer Biology & Therapy

Article Title: Antibody dependent cell-mediated cytotoxicity selection pressure induces diverse mechanisms of resistance

doi: 10.1080/15384047.2023.2269637

Figure Lengend Snippet: STAT1 signaling is associated with but does not drive acquisition of ADCC resistance (a), western blot analysis of STAT1 pathway proteins in ADCC-sensitive (ADCCS) and ADCC-resistant (ADCCR) A431, SKOV3, and FaDu cells. Densitometry values for expression normalized to GAPDH in ADCC-resistant relative to ADCC- sensitive cells are indicated. (b), percent cytotoxicity of ADCC-sensitive (blue) and ADCC-resistant (red) A431 cells as measured by ADCC assay when cells are incubated in either medium alone (solid bars) or medium supplemented with DMSO (horizontal hash bars) or 10 nM ruxolitinib (diagonal hash bars) for 72hrs prior to ADCC assay ( n = 3). Unpaired two-tailed t -test, ns, not significant. Error bars, SEM. (c), Representative western blot analysis of phospho-STAT1 protein expression in ADCC-sensitive and ADCC-resistant A431 cells when cells are incubated in either medium alone (untreated) or medium supplemented with DMSO or 10 nM ruxolitinib for 72hrs. Densitometry values for expression normalized to GAPDH in cells treated with DMSO or ruxolitinib compared to untreated cells are indicated. (d), percent cytotoxicity of ADCC-sensitive (blue) and ADCC-resistant (red) A431 cells as measured by ADCC assay when cells are incubated in either medium alone (solid bars) or transfected with control scramble siRNA (siNEG, horizontal hash bars) or siRNA specific for STAT1 (diagonal hash bars) for 48 h prior to ADCC assay ( n = 3). Unpaired two-tailed t -test, **, P <.01. Error bars, SEM. (e), Representative western blot analysis of STAT1 protein expression in ADCC-sensitive and ADCC-resistant A431 cells when cells are incubated in either medium alone (untreated) or transfected with control scramble siRNA (siNEG) or siRNA specific for STAT1 for 48hrs. Densitometry values for expression normalized to GAPDH in cells transfected with control scramble (siNEG) or siRNA specific for STAT1 compared to untreated cells are indicated. (f), western blot analysis of STAT1 protein expression in untreated and ADCC condition treated parental A431 cells, A431 cells transduced with a non-targeting control CRISPR-cas9 (NTC), and A431 cells transduced with CRISPR-cas9 specific for STAT1 (STAT1-KO) at the indicated challenges. (g), percent cytotoxicity of untreated and ADCC condition treated parental A431 cells, A431 cells transduced with a non-targeting control CRISPR-cas9 (NTC), and A431 cells transduced with CRISPR-cas9 specific for STAT1 (STAT1-KO) at every 5 challenges during derivation of resistance as measured by ADCC assay ( n = 2). Unpaired two-tailed t- test, *, P <.05. Error bars, SEM.

Article Snippet: Western blots were conducted with Cell Signaling antibodies to: E-Cadherin (3195), Vimentin (5741), Snail (3879), Twist (46702), EGFR (4267), pEGFR Y1068 (3777), pEGFR Y1045 (2237), HER2 (4290), pHER2 Y1248 (2247), ERK1/2 (4695), pERK1/2 T202/Y204 (4370), AKT (pan) (4691), pAKT S473 (4060), CD54 (65055), STAT1 (14994), pSTAT1 Y701 (9167), M×1(37849), IL-6 (12153), ATR (2790), pCHK1 S345 (2341), PSMB8 (13635), and PSMB10 (78385) at concentrations of 1:1000 diluted in 5% milk in TBST.

Techniques: Western Blot, Expressing, ADCC Assay, Incubation, Two Tailed Test, Transfection, Transduction, CRISPR